Method of producing elastases by bacteria

ABSTRACT

Some strains of Flavobacterium produce highly active elastase. The elastase is useful as a meat tenderizer.

United States Patent [191 Shiio et al.

[ Nov. 12, 1974 i METHOD OF PRODUCING ELASTASES BY BACTERIA [75] Inventors: lsamu Shiio; Hachiro Ozaki; Tsuyoshi Nakamatsu, all of Kanagawa, Japan [30] Foreign Application Priority Data Dec. 14, 1971 Japan 47-2719 [52] US. Cl. 195/65 [51] Int. Cl Cl2d 13/10 [58] Field of Search 195/65, 66 R [56] References Cited OTHER PUBLICATIONS Morihara et al., Archives of Biochemistry & Biophysics, Vol. 120, pages 6878 (1967),

Primary E.\'aminerLi0nel M. Shapiro Attorney, Agent, or FirmHans Berman; Kurt Kelman [57] ABSTRACT Some strains of Flavobacterium produce highly active elastase. The elastase is useful as a meat tenderizcr.

2 Claims, N0 Drawings METHOD OF PRODUCING ELASTASES BY voges-Pwskauer reacting;

. egative. I Y. H25: Produced. The present invention relates to the preparation of gt rph: syt irolgze ltrrc acl ti ize dastases ba Pigment Produced. Elastase decomposes and converts ClflStlItWhlCh 1s a 5 E5522; scleroprotein into watersoluble protein, and is useful Aerobic as a meat tenderizer and as a depilatory in the fur in- O-F test: Metabolizedby oxidation. dustries Assimilation of carbohydrates:

7. v Acid produced from glucose, fructose,

It is known that microorganisms belonging to genera l0 l to se, rgh, mannojse agdf Bacillus, Staphylococcus,Flavobacterium, Pseudomo- 335?' y gfi gg f yg mm has and Streptomyces produce elastase, however, the galactolseljactose. mannitol.

. inosito an sorhitol. elastase activity of the known elastases is very low. Cellulose is decomposm It ha nowbeenfounsl. a n w ains o v ba terium produce highly active elastase in a very large amount m the culturebroth.

Bacteria whichproduce the elastase includeFlavo- .rn ol m Growth bacterium immotum No. 935 (FERM P-l308yMicro- P mdmm" 37 C organisms ldfintlfled byii F-ERMiP- numbers .are avatl- LHavobacterium Negafive Negative NO growth able to the public fromthe Fermentatron Research Inaquatile stitute, Agency of lndustrialScience and Technology, ng gaf a Ncgflwe Posmve Mmlme of the Ministry forlndustrialTrade andlndustry, Ja- MA 945 i i Negative Modemc pan), Flavobacterium'incertum No. 84-3 (FERM 'P- g -E24 y gg Swi m f 1309) and Flavobacterium pulchlum No. 87-5 (PERM 7 we ega we 0 P-13-t0). 25

The characteristic properties of "Flavobacterium Stmt vcomlmnson P 'j lchayractenstlcs of the PERM P4308F1av0bacterium @FERM P4309 and strains-was carried out with Bergeys'Manual of Deterplavobacteriumf M .p 13 oo i n minativeBacteriology, 7th edition, and the strains are considered to belong to ,genus- Flavobacterium. Their L Morphological properties: .30 properties did-not agree with Flavobacterlum aquatzle Cells: Roda-blunt, 0.9-1.1 x-2.0 4.0 microns. and-Flavobactertum fucatum, whlch have many com- Mobility: Non-motile. u spore: NotpmducedflMimcystnotpmduced mon properties w th the present strarnsas shown .m 3 Gram stein: Negative. Table .l. Thesstrarns,accordingly, are considered to Growth media represent new species fortwhichthe names F lav0bacter- Bouillon agar plate: Growth moderate,-smooth, circular,

entire o,ange. ye"owish and vmm tmm0tum,'Flav0bactermm mcertum and Flavobac- B r r gg sg nfip teriumpulc'hlum areproposed. 835 32 :2; stab: gg gf pe e As can be seenffrom the'following Table.2, F lavobac- Litmus milk: Alkaline, peptonized. .terium immotum, Nou9-35, 'Flavo'bacterium incerlum $gf-gl" Negative. N0. and'Flavobacterium;pulchlumNo. 87-5 prorMethylredtgst; Negative. 40 duce elastases in very high-yield.

Table '2 Amount of elastase-produced (units/ml) in Strain Medium 1 Medium" Medium lll Flavobacterium l 10 75 66 immotum 9-35 Flavobacterium l l l 82 68 incertum 84-3 Flavobacterium 7O 44 pulchlum 8.7-5 Flavobacterium 8.6 0 H0 kawanense'lAM l0l4 Flavobacterium 8.0 0 0 suaveolens'lAM ll3l Flavobacterium 9.8 0 0 arborescens [AM H00 .Flavobacterium 8.0 0 0 citreum MM 1158 Flavobacterium 8.0 O 0 fuscum lAM ll8l Flavobacterium 7.2 0 0 rignese lAM i238 Flavobacterium 9.0 0 O sulfureum [AM 1252 Flavobacterium 4.0 0 0 ferrugineum lAM l493 Flavobacterium 8.4 O 0 aurantianum CCM 73 Flavobacterium 8.0 0 O flavescens'CCM 1079 Flavobacterium 3:5 0 0 rhenanum CCM 298 Fluvohactcrium 8.1 (l 0 Table 2 Continued Amount of elastase produced (units/ml) in aeruginosa [F 3455 Notes:

1. Elastase activity was determined for supernatant liquids from 24 hours culture broths.

2. 1 unit/ml of elastase activity is that of one milligram of elastin dissolved in one milliliter culture medium, when incubated at 40 C at pH 7.2 for 1 hour in 0.025M Tris-HCl buffer.

3. Composition of media I, II and 111 Medium I: 3 percent Casein, 4 percent glucose, 0.2 percent corn steep liquor, 0.1 percent K HPO and 0.01 percent MgS04, PH 7.0.

Medium 11: 0.5 percent Elastin, 1.5 percent Polypeptone, 1 percent yeast extract, 0.5 percent glucose, 0.1 percent K HPO and 0.01 percent MgSO4, PH 7.0.

Medium Ill: 1 percent Meat extract, 1 percent Polypeptone," 1 percent yeast extract, 0.5 percent glucose and 0.5 percent NaCl, pH 7.0.

The bacteria of the invention are cultured on a medium containing an assimilable carbon source, as assimilable nitrogen source, inorganic salts, organic nutrients and growth promoting substances. The assimilable carbon sources include carbohydrates such as glucose, sucrose, starch hydrolyzate, molasses and sorbitol; organid acids, such as acetic acid, propionic acid and a-ketoglutaric acid; alcohols, such as ethanol and propanol; and hydrocarbon for selected strains. The assimilable nitrogen sources include inorganic nitrogen compounds, and natural organic substances, such as protein, its hydrolyzate and amino acids. The cultivation is carried out at a pH between 5 and 9 under aerobic conditions.

Elastase produced in the culture broth may be isolated by conventional methods, such as by salting out with ammonium sulfate, by chromatography using ion exchange resin, Sephadex or ion exchange cellulose, or by precipitation with acetone or alcohol.

Elastase activity was estimated by the colorimetric method of Sacker et al (L. A. Sacker et al; Proc. Soc. Exp. Bio]. and Med. 90, 323, i955), i.e., milligrams orcein elastin were digested in 2 ml of 0.025 M Trisbuffer for 60 minutes at 40 C at pH 7.2 with shaking.

Non-specific protease activity was determined by Lowrys method (Lowry O. H. et al; Journal of Biological Chemistry, 193 265, 1951).

The elastin used for determining elastase activity was prepared from ligamentum nuclae of cattle by Partridges method (Partridge S. M. et a1; Biochemical Journal 61, 11, 1955).

Elastases obtained by the present invention are highly effective in hydrolyzing elastin, although their properties slightly vary with the strain used.

The elastase produced by Flavobacterium immotum No. 9-35 has the following properties:

1. The elastase activity is about 18 times that of crystalline papain (manufactured by Sigma Co.).

2. The casein digesting activity is almost the'same as that of the crystalline papain.

3. The optimum temperature is at 40 C.

4. The optimum pH is at 79.

5. The elastase is inactivated with cupric and zinc ions.

6. Heat stability 200 Micrograms of the elastase were dissolved in l milliliter of 0.025M Tris-buffer solution, incubated at pH 7.2 at various temperatures for 5, 20 or 60 minutes, whereupon its residual activity was determined, and is listed in Table 3.

pH 2 Sodium citrate-HCI buffer pH 3-6 Sodium citrate-citric acid buffer pH 7 2-(N-Morpholino)ethunesulfonic ncid-NuOH buffer pH 8-9 Tris-HCl buffer pH 10-11 Ammonium chloride-ammonium hydroxide buffer 8. Effect of metal ions and SH reagents or elastase activity Table inhibitor (1 mM) Relative Activity None 100 NaCl 100 SnCl; 100 MgSO, 98 LiSO, 100 FeSO, 100 BaCl, 97 MnSO, 80 CaCl- 73 CoCl 72 CuSO, 0 ZnSO, 0 HgCl 0 CMB 110 DTT 38 Glutathione 100 L-Cysteine 100 EDTA 100 CuSOyl-EDTA 40 ZnSO +EDTA 40 DTT CMB 74 CMB: p-Chloromercuribenzoate DTT: Dithiolhreitol EDTA: Ethylcnediaminetetraacetate EXAMPLE 1 1 liter culture medium containing;

Polypeptone 1.5 Elastin 0.5 Yeast extract 1.0 Glucose 0.5 KH PO 0.05 K i-1P0, 0.1 MgSO .7H O 0.01

was prepared, inoculated with Flavobacterium immotum No. 9-35 (FERM P-1308), and cultured at 30 rial cells, and the supernatant liquid was found to contain 91 units per milliliter of elastase. Ammonium sulfate was added to the supernatant liquid to give 0.45 saturation. Precipitate formed and was collected by centrifugation, dissolved in water, and dialyzed in tap water for 2 hours, and acetone was added to the dialyzed solution. The precipitate formed in percent acetone solution was collected, and dried in vacuo to yield 322 mg crude enzyme powder. 1 milligram of the powder solubilized 214 mg elastin when incubated at 40 C for 1 hour.

EXAMPLE 2 1 liter culture medium containing 3 percent casein, 4 percent glucose, 0.2 percent corn steep liquor, 01

percent Kl-l PO 0.2 percent K l-lPO and 0.01 percent MgSO 7H O, of pH 7.0 was prepared, inoculated with Flavobaclerium immotum No. 9-35 (PERM p-l308), and cultured at 30 C for 24 hours with aerating. The culture broth was found to contain 137 units/ml of elastase. The culture broth was worked up in the same way as described in Example 1, and 900 mg of crude en-' zyme powder were obtained. 1 milligram of the powder solubilized 114 mg elastin at 40 C in 1 hour.

EXAMPLE 3 of the powder solubilized mg of elastin at 40 C in 1 hour.

EXAMPLE 4 Flavobacterium pulchlum No. 87-5 (FERM P-l3l0) was cultured in the same way as in Example 2, and the culture broth was found to contain 70 units/ml of elastase.

What we claim is:

l. A method of producing elastase which comprises culturing an elastase-producing strain of Flavobacterium immotum, Flavobacterium incertum, or Flavobacterium pulchlum on an aqueous nutrient medium containing assimilable sources of carbon and nitrogen, inorganic salts and minor organic nutrients until elastase accumulates in said medium, and recovering the accumulated elastase.

2. A method as set forth in claim 1, wherein said bacterium is F lavobacterium immotum FERM P-1308, F lavobaclerium incertum PERM P-1309 or F lavobacterium pulchlum Ferm P-13l0. 

1. A METHOD OF PRODUCING ELASTASE WHICH COMPRISES CULTURING AN ELASTASE-PRODUCING STRAIN OF FLAVOBACTERIUM IMMOTUM, FLAVOBACTERIUM INCERTUM, OR FLAVOBACTERIUM PULCLUM ON AN AQUEOUS NUTRIENT MEDIUM CONTAINING ASSIMILABLE SOURCES OF CARBON AND NITROGEN, INORGANIC SALTS AND MINOR ORGANIC NUTRIENTS UNTIL ELASTASE ACCUMULATES IN SAID MEDIUM, AND RECOVERING THE ACCUMULATED ELASTASE.
 2. A method as set forth in claim 1, wherein said bacterium is Flavobacterium immotum FERM P-1308, Flavobacterium incertum FERM P-1309 or Flavobacterium pulchlum Ferm P-1310. 